Mollusks of the genus Conus produce a highly toxic venom which enables them to carry out their unique predatory lifestyle. Prey are immobilized by the venom which is injected by means of a highly specialized venom apparatus, a disposable hollow tooth which functions both in the manner of a harpoon and a hypodermic needle.
Few interactions between organisms are more striking than those between a venomous animal and its envenomated victim. Venom may be used as a primary weapon to capture prey or as a defense mechanism. These venoms disrupt essential organ systems in the envenomated animal, and many of these venoms contain molecules directed to receptors and ion channels of neuromuscular systems.
The predatory cone snails (Conus) have developed a unique biological strategy. Their venom contains relatively small peptides that are targeted to various neuromuscular receptors and may be equivalent in their pharmacological diversity to the alkaloids of plants or secondary metabolites of microorganisms. Many of these peptides are among the smallest nucleic acid-encoded translation products having defined conformations, and as such they are somewhat unusual. Peptides in this size range normally equilibrate among many conformations, and proteins having a fixed conformation are generally much larger.
The cone snails that produce these toxic peptides, which are generally referred to as conotoxins or conotoxin peptides, are a large genus of venomous gastropods comprising approximately 500 species. All cone snail species are predators that inject venom to capture prey, and the spectrum of animals that the genus as a whole can envenomate is broad. A wide variety of hunting strategies are used; however, every Conus species uses fundamentally the same basic pattern of envenomation.
The major paralytic peptides in these fish-hunting cone venoms were the first to be identified and characterized. In C. geographus venom, three classes of disulfide-rich peptides were found: the .alpha.-conotoxins (which target and block the nicotinic acetylcholine receptors); the .mu.-conotoxins (which target and block the skeletal muscle Na.sup.+ channels); and the .omega.-conotoxins (which target and block the presynaptic neuronal Ca.sup.2+ channels). However, there are multiple homologs in each toxin class; for example, at least five different .omega.-conotoxins are present in C. geographus venom alone. Considerable variation in sequence is evident, and when different .omega.-conotoxin sequences were first compared, only the cysteine residues that are involved in disulfide bonding and one glycine residue were found to be invariant. Another class of conotoxins found in C. geographus venom is that referred to as the conantokins which cause sleep in young mice and hyperactivity in older mice and are targeted to the NMDA receptor. Each cone venom appears to have its own distinctive group or signature of different conotoxin sequences.
Many of these peptides have now become fairly standard research tools in neuroscience. The .mu.-conotoxins, because of their ability to preferentially block muscle but not axonal Na.sup.+ channels, are convenient tools for immobilizing skeletal muscle without affecting axonal or synaptic events. U.S. Pat. No. 5,432,155 discloses a group of bioactive conotoxin peptides which are extremely potent inhibitors of synaptic transmission at the neuromuscular junction and/or which are targeted to specific ion channels. Many of them appear to be members of the known class of .mu.-conotoxins.
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels which are key components of nervous systems. The classical role for these receptors was defined at the neuromuscular junction: nicotinic receptors concentrated on the muscle end plate serve as the key macromolecules that detect release of a neurotransmitter from the presynaptic terminus of the motor axon. However, in addition to these skeletal muscle nicotinic receptors, many other molecular forms of nicotinic receptors exist; these are generally referred to as neuronal nAChRs.
The .omega.-conotoxins have become standard pharmacological reagents for investigating voltage-sensitive Ca.sup.2+ channels and are used to block presynaptic termini and neurotransmitter release. The .omega.-conotoxin GVIA from C. geographus venom, which binds to neuronal voltage-sensitive Ca.sup.2+ channels, is an example of such. The affinity (K.sub.d) of .omega.-conotoxin GVIA for its high-affinity targets is sub-picomolar; it takes more than 7 hours for 50% of the peptide to dissociate. Thus the peptide can be used to block synaptic transmission virtually irreversibly because it inhibits presynaptic Ca.sup.2+ channels. However, .omega.-conotoxin is highly tissue-specific. In contrast to the standard Ca.sup.2+ channel-blocking drugs (e.g. the dihydropyridines, such as nifedipene and nitrendipene, which are widely used for angina and cardiac problems), which can bind Ca.sup.2+ channels in smooth, skeletal, and cardiac muscle as well as neuronal tissue, .omega.-conotoxins generally bind only to a subset of neuronal Ca.sup.2+ channels, primarily of the N subtype. The discrimination ratio for .omega.-conotoxin binding to voltage-sensitive Ca.sup.2+ channels in neuronal versus nonneuronal tissue (e.g. skeletal or cardiac muscle) is greater than 10.sup.8 in many cases.
The .alpha.-conotoxins have various clinical uses. One such use is their utility as clinical muscle relaxants because of their ability to achieve antagonistic blockage of the mammalian neuromuscular junction nAChRs. Another use for the .alpha.-conotoxins is in the diagnosis of myasthenia gravis. Some .alpha.-conotoxins have been found to bind preferentially to neuronal nicotinic receptors rather than to neuromuscular receptors.
Examples of such peptides are shown in U.S. Pat. Nos. 5,432,155; 5,595,972 and 5,633,347, the disclosures of which are incorporated herein by reference.
Additional conotoxin peptides having these general properties continue to be sought.